Liquid Chromatography-Mass Spectrometry (LC-MS).pptx

Liquid Chromatography-Mass Spectrometry (LC-MS)

1. LIQUID CHROMATOGRAPHY- MASS SPECTROMETRY Subject- Methods in Pharmaceutical Research DEPARTMENT OF PHARMACEUTICAL SCIENCES Dr. Hari Singh Gour Vishwavidyalaya Sagar (M.P)- 470003, India (A Central University) Submitted By: DEBASIS SEN M.PHARM 1 ST SEMESTER Y22254008 Submitted To: Prof. A. GAJBHIYA (Professor, DOPS) Prof. S. K. KASHAW (Professor, DOPS)

2. CONTENT WHAT IS HYPHENATED TECHNIQUE INTRODUCTION TO LC-MS PRINCIPLE PROBLEMS IN COMBINING HPLC AND MS WORKING OF LC-MS INTERFACE MASS ANALYZER DETECTORS CONCLUSION REFERENCES

3. What is Hyphenated Technique? Hyphenated Technique: The term “hyphenation” was first adapted by Hirschfeld in 1980. The technique developed from the coupling of a separation technique and spectroscopic detection technology is known as hyphenated technique. Advantages of hyphenated techniques: Fast and accurate analysis. Higher degree of automation. Reduction of contamination due to its closed system. Separation and quantification achieved at same time.

4. INTRODUCTION TO LC-MS Liquid Chromatography/Mass Spectrometry (LC/MS) is a powerful analytical technique that combines the resolving power of liquid chromatography with the detection specificity of mass spectrometry. Liquid chromatography (LC) separates the sample components and then introduce them to the mass spectrometer (MS). The MS creates and detects charged ions. The LC/MS data may used to provide information about the molecular weight, structure, identity and quantity of specific sample components. In LC-MS we remove the detector from the column of LC and fitting the column to interface of MS. In the most of the cases the interface used in LC-MS are ionization source.

5. PRINCIPLE The LC-MS technology involves use of an HPLC, wherein the individual components in a mixture are first separated followed by ionization and separation of the ions on the basis of their mass/charge ration. The separated ions are then directed to a electron multiplier tube detector, which identifies and quantifies each ion. The ion source is an important component in any MS analysis, as this basically aids in efficient generation of ions for analysis. To ionize intact molecules, the ion source could be APCI (Atmospheric Pressure Chemical Ionization), ESI (Electron Spray Ionization) etc.

6. PROBLEMS IN COMBINING HPLC AND MS HPLC MS Liquid phase operation Vacuum operation 20-50 °C 200-300 °C No mass range limitation Up to 4000 Da for quadrupole MS Inorganic buffer Requires volatile buffers 1ml/min eluent flow is equivalent to 500ml/min in gas Accept 10ml/min gas flow

7. WORKING OF LC/MS LC-MS is mainly separated into the three parts- chromatography, interface and mass spectrometry. In liquid chromatography, separation is performed and the separated components transferred to the interface. In interface the liquid is volatilized and transferred to the MS. With the various ionization technique the compound is ionized and then analysed by mass analyser.

8. INTERFACES LC-MS systems include a device for introducing samples such as HPLC, an interface for connecting such device, an ion source that ionizes samples, an electrostatic lens that efficiently introduces the generated ions, a mass analyzer unit that separates ions based on their mass-to-charge (m/z) ratio, and a detector unit that detects the separated ions. In a LC-MS system, however, if the LC unit is simply connected directly to the MS unit, the liquid mobile phase would vaporize, resulting in large amounts of gas being introduced into the MS unit. This would decrease the vacuum level and prevent the target ions from reaching the detector. So, the interfaces are to be used.

9. TYPES OF INTERFACES It is difficult to interface a liquid chromatography to a mass spectrometer cause of the necessity to remove the solvent. The commonly used interfaces are- Electrospray Ionization (ESI) Atmospheric Pressure Chemical Ionization (APCI) Atmospheric Pressure Photoionization (APPI) Thermospray Ionization (TSI)

10. ELECTROSPRAY IONIZATION It is a type of evaporative ionization technique used to analyze the high molecular weight biomolecules, labile and non-volatile compounds. In ESI, the sample is dissolved in a volatile solvent, typically a mixture of water and an organic solvent such as methanol or acetonitrile. The sample solution is then introduced into a small-diameter capillary held at a high voltage, typically 2-5 kV, which generates a strong electric field at the capillary tip. The electric field causes the solvent to evaporate, leaving behind a charged droplet containing the sample molecules. As the droplet moves towards the mass analyzer, it becomes increasingly charged, causing it to break up into smaller droplets. The smaller droplets carry some of the sample molecules, which become further ionized by the electric field. This process is repeated until the droplets become small enough to produce individual gas-phase ions.

11. The ions produced by ESI are typically multiply charged, due to the presence of multiple charged species in the droplet, and are often detected in the positive ion mode. The ions produced by ESI are typically analyzed by a mass analyzer, which separates the ions based on their mass-to-charge ratio (m/z). The resulting mass spectrum provides information about the mass and composition of the sample molecules.

12. ATMOSPHERIC PRESSURE CHEMICAL IONIZATION (APCI) APCI is a type of soft ionization technique based on the mechanism of evaporation and carried out at atmospheric pressure. APCI vaporize solvent and sample molecules by spraying the sample solution into a heater ( heated to about 400C) using a gas, such as N2. Solvent molecules are ionized by corona discharge to generate stable reaction ions.

13. ATMOSPHERIC PRESSURE PHOTOIONIZATION (APPI) The LC eluent is vaporized using a heater at atmospheric pressure. The resulting gas is made to pass through a beam of photons generated by a discharge lamp (UV lamp) which ionizes the gas molecules.

14. THERMOSPRAY IONIZATION (TSI) It is a evaporative ionization method in which solvent molecules are removed from the analyte by evaporation. Eluent sample coming from the column will be passed through the heated capillary tube. Heated capillary tube will nebulize the eluent, partially evaporate the solvent and form a stream of fine spray containing analyte. Fine spray droplets will be partially charged by thermal energy and solvent gets evaporated from spray. Ionized analytes will move towards the mass analyser for further analysis.

15. MASS ANALYZER They deflect ions down a curved tubes in a magnetic fields based on their kinetic energy determined by the mass, charge and velocity. The magnetic field is scanned to measure different ions. Types of mass analyser- Quadrupole mass filter Time of flight Ion trap

16. QUADRUPOLE MASS ANALYZER It consist of four cylindrical metal rods arranged in a square parallel to the direction of ion beam and radio frequency (RF) or direct current (DC) voltage is applied. Ions will enter into the mass analyser and depending on the ratio of RF amplitude and DC voltage oscillating electrostatic filed will be generated for ions. If RF>DC, then larger ion will hit the detector first If RF<DC, then small ions will hit the detector first. Inappropriate m/z ratio of ions (other than 1-1000) will undergo unstable oscillation and hit the rod, and ultimately will not reach the detector.

17. TIME OF FLIGHT (TOF) MASS ANALYZER ToF is based on the simple idea that the velocities of two ions varies depending on the mass of the ion but the ions should have created at same constant and should have same kinetic energy. Light ion will strike the detector first due to higher velocity.

18. ION TRAP MASS ANALYZER Ion trap mass analyser is a high resolution, high sensitivity and multiple product ion scan capability. One end cap electrode has single small central aperture through which ions are introduced into trap while other one has several apertures through which ions are passed to a detector. Helium bath gas is present in the trap to stabilize the ion trajectories. Collison takes place between helium bath gas and ions. Due to this motion of ions increases the trapping efficiency of the analyser. The ions are ejected from the trap on the basis of m/z values to create mass spectrum.

19. DETECTORS The detector is an important tool of mass spectrometer that produces current that is proportional to the number of ions strike it. Once the ions are formed passed from analyser they have to be detected and transformed into signal. Commonly used detectors-

20. CONCLUSION The LC-MS is a hyphenated technique used in combination with separation power of HPLC with detection power of Mass spectrometry. It is widely used in pharmaceutical, chemical, food, agrochemical industries, environmental and forensic applications. LC-MS is used for qualitative and quantitative determination of drug substances and biological samples. Also it is commonly used in drug research and quality control.

21. REFERENCES Alhendi AS. A review: Protein identification by LC-MS: Principles, instrumentation, and applications. Iraqi J Sci. 2020;61(10):2428-66. Kumar PR, Dinesh SR, Rini R. LCMS—a review and a recent update. J Pharm Pharm Sci. 2016 Mar 1;5:377-91. Korfmacher WA. Foundation review: Principles and applications of LC-MS in new drug discovery. Drug discovery today. 2005 Oct 15;10(20):1357-67.

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